Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022

Background Vancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible. Aim The aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE. Methods From 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance. Results In total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm. Conclusion From 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.


Background
Enterococcus faecalis and Enterococcus faecium can cause urinary tract infections, intra-abdominal infections, skin infections and bloodstream infections [1].Enterococcus faecium belongs to the ESKAPE bacteria, a group of six pathogens which includes Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.besides E. faecium [2].They can be difficult to treat since they are intrinsically resistant to many antibiotics such as cephalosporins, trimethoprim-sulfamethoxazole and lincosamides [3].Furthermore, E. faecium can acquire antibiotic resistance through chromosomal mutations or gene exchange.High-level resistance to aminoglycosides and resistance to ampicillin and glycopeptides are frequent in E. faecium [4].Many E. faecium are multidrug-resistant, and they are a leading cause of nosocomial infections worldwide [5].During the last 10 years, vancomycin-resistant E. faecium (VREfm) have increased in several countries in Europe [6].Eight types of acquired vancomycin resistance genes, vanA, vanB, vanD, vanE, vanG, vanL, vanM and vanN, have been reported [7].In Europe, vanA and vanB are most prevalent among clinical VREfm isolates.Vancomycinvariable enterococci (VVE) are E. faecium that have the vanA gene complex but are phenotypically vancomycin-susceptible [8].In the laboratory, VVE can only be detected by molecular methods and cannot be cultured on selective vancomycin-containing medium.They are capable of shifting from a vancomycin-susceptible phenotype to a resistant phenotype during vancomycin therapy in patients, thus limiting the success of treatment and representing an important source for vancomycin resistance genes [9].
In general, if VRE are highly resistant to other antimicrobial agents, the two major remaining antibiotics are linezolid and daptomycin [10].Resistance to linezolid in enterococci is often due to mutations in the V domain of the 23S rRNA gene (G2576T or G2505A).Furthermore, transferable resistance genes encoding linezolid resistance in Enterococcus spp., cfr, cfr(B), optrA and poxtA, have been described.A web tool, LRE-Finder, has been developed for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences [11].To our knowledge, no web tools have been developed for detection of daptomycin resistance or tigecycline from whole genome sequencing (WGS) data.
We have previously described the surveillance of VRE and VVE in clinical isolates in Denmark from 2005 to first quarter of 2019 [12,13].In the present study, we describe van genes detected in the VRE and VVE isolated from 2015 to 2022.We investigated whether major E. faecium clusters were present both at regional and national level.Furthermore, we tested VRE and VVE isolates for mutations and genes encoding linezolid resistance.

Healthcare regions
Denmark is divided into five geographical and administrative regions (Figure 1): Capital Region of Denmark (1,867,948

Vancomycin-resistant and vancomycin-variable enterococcal isolates
In Denmark, VRE are not notifiable.The 10 DCMs receive samples from hospitals and general practitioners in the five regions.They isolate Enterococcus spp.from different clinical samples, e.g.urine, blood and tissue.Vancomycin resistance is detected using a phenotypical method according to EUCAST [15].Furthermore, VVE are detected by PCR of E. faecium isolates.Since 2015, the 10 DCMs have voluntarily submitted to the National Reference Laboratory for Antimicrobial Resistance at Statens Serum Institut (NRL/SSI) the VRE and VVE isolates from clinical samples, but not (faecal) isolates from screening [12].
We included in this study only one VRE and VVE isolate per patient within a 12-month period, except if both an E. faecalis and an E. faecium were detected from the same patient within the same year.The personal identification number was used as identifier, so even when a patient with VRE was moved to another hospital, only one VRE was included.
To determine any under-reporting in the submissions, we compared the number of VRE and VVE submitted to SSI with data from clinical VRE reported by the DCMs to the Danish Microbiology DataBase (MiBa).
The MiBa database serves both as a tool for clinical doctors to access clinical microbiology test results on their patients and a national surveillance and research database [16].Data from microbiological analyses performed by the DCMs are registered in MiBa.Each test report in MiBa contains information on the individual case (personal identification number, age at sample, sex), the sample (sample ID, sampling date and time, specimen type (blood, urine, etc)), the diagnostic analyses performed, and results including microbiological species, phenotypic antimicrobial susceptibility and in some cases resistance genes (e.g.vanA and vanB) [17].

Whole genome sequencing typing
From 2015 through 2022, all clinical VRE and VVE isolates (n = 4,090) underwent WGS.Genomic DNA was extracted (DNeasy Blood and Tissue Kit, Qiagen), with subsequent library construction (Nextera Kit, Illumina), and WGS was performed (Nextseq, Illumina) to obtain paired-end reads of 2 × 150 bp.The quality and quantity of the raw reads of all isolates were assessed using the Bifrost QC pipeline and assembled into draft genomes using the SKESA assembler version 2.2.Quality control, detection of resistance genes and multilocus sequence types (MLST) as well as species identification was carried out using the Bifrost QC pipeline using the MLST and ResFinder databases available at the Center of Genomic Epidemiology homepage (www.cge.food.dtu.dk).The isolates were further subtyped in SeqSphere+ version 8.5.1 (Ridom GmbH) using the E. faecium cgMLST scheme by de Been et al. [18].Local single linkage clustering (SLC) identification numbers were defined for E. faecium isolates, clustering isolates with up to 20 allellic distances, sometimes including multiple complex types.The local SLC clusters were named after the complex type of the earliest observed E. faecium isolate within each cluster [18].
Furthermore, we tested all VRE and VVE isolates from 2015 to 2022 for the mutations and genes encoding linezolid resistance using the LRE-Finder.The LRE-Finder detects the fraction of thymine bases in position 2,576 and the fraction of adenine bases in position 2,505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment [11].

Analysis related to typing
From 2015 to 2022, a total of 4,090 VRE and VVE were sent to SSI.Most of the samples were isolated from urine (65%), 11% were from blood, and the rest were from other clinical specimen (e.g.wounds, tracheal secretions or pus from the abdomen).In total,

Linezolid-and vancomycin-resistant enterococci
We investigated WGS data from the VRE and VVE isolates for 23S rRNA mutations and optrA, cfr, cfr(B) and poxtA genes using the LRE-Finder.Linezolid resistance was encoded by the G2576T 23S rRNA mutation in the LVVEfm isolates and was also often most often detected among the LVREfm isolates (Table ).

Discussion
The numbers of VRE and VVE in Denmark increased from 520 isolates in 2015 to 827 isolates in 2022.In total, 19% of the isolates were not sent to SSI, but detected from MiBa.Those 19% were distributed evenly throughout the study period and it did not seem to be a systematic loss.Since these MiBa isolates not were typed, it is hard to know which van genes and cgMLST they had.From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium.During 2020, a shift started from vanA E. faecium to vanB E. faecium.A similar shift was detected in the end of the 2000s in Germany [19].In 2020, vanB-positive E. faecium were still dominating in Germany [20].Also in the Netherlands, a shift occurred from vanA E. faecium outbreaks in 1999 to 2014 to vanB E. faecium outbreaks between 2014 and 2017 [21].Until 2019, vanB E. faecium were more prevalent than vanA E. faecium in Norway [22].
The number of VRE differed between the Danish regions, which can partly be explained by different laboratory methods used in different DCMs but also by differences in the type of specimen tested for VRE.One DCM in the Region of Southern Denmark did not test enterococci from urine for vancomycin susceptibility (data not shown).As VRE are most often detected from urine on a national basis, a large number of VRE might be missing from this DCM.As this DCM had overlap in patients with other hospitals in the region, we would expect that the distribution of van genes and genetic clusters would be the same as the tested VRE isolates from the rest of the region.
The use of molecular diagnostics to detect VVE influences the detection of VVE.Since the emergence of the VVE cluster ST1421-CT1134, most DCMs have been using vanA PCR on all invasive isolates, some also on all clinical isolates.The difference from DCM to DCM in the use of vanA PCR for detection of VVE is a bias, but it is hard to know how big this bias is.
There are national guidelines on infection prevention and control measures for VRE, but VRE are not notifiable in Denmark [24].Therefore, the infection prevention  From 2019 to 2022, an increase in VRE was detected for the Zealand Region, Central Denmark Region and North Denmark Region.This a higher workload for hospital staff in charge of patient isolation and outbreak investigations.We did not examine the economic cost associated with VRE infections in healthcare, but an increase in the number of VRE can be expected to have financial implications [25].
During 2012 and 2013, ST80-CT14 vanA E. faecium was highly prevalent in the Capital Region.This is similar to the Group2_ST80 in the study by Pinholt et al. analysing Danish data from January 2012 to April 2013 [26].ST80-CT14 vanA E. faecium occurred in all Danish Regions during 2015.To our knowledge, ST80-CT14 vanA E. faecium has not been reported outside Denmark, so the origin of this cluster is unknown.
ST117-CT24 vanA E. faecium was first detected in the Capital Region in January 2015; its origin is unknown.Since the late 1990s, ST117-CT24 vanA E. faecium has spread in several regions in Germany and was also detected in the Netherlands in 2014 [19,27].Furthermore, an Irish/Danish study detected ST1478-CT24 (a single locus variant of ST117) VREfm from Ireland, with 15 allelic differences to the nearest Danish ST117-CT24 VREfm isolate [28].It therefore seems likely that the Danish ST117-CT24 VREfm isolate was imported to Denmark from another European country.[36].This shows that the cluster has endemic potential.A clone-specific PCR which can detect ST117-CT36 vanB E. faecium was used in the Capital region.One could speculate that this may be the reason why this cluster was not as successful as others, because this particular PCR allowed fast identification and elimination of this cluster.
The first report of ST80-CT2406 E. faecium in the cgMLST E. faecium database was during 2019.In August 2019, the first detection of ST80-CT2406 vanB E. faecium was a screening sample from a patient transferred from a German hospital before detection (personal communication: Mette Pinholt, August 2023).
We found spread of seven clusters during 8 years.
In some cases, the same clusters were seen abroad and in two cases, the high epidemic clusters were imported.A global dissemination of hospital clones of E. faecium has also been described [37].This emphasises the need to test for VRE if the patient had been hospitalised abroad.
We do not know why these seven E. faecium clusters were more successful than other VREfm types.It can be investigated whether these clones have more potential virulence genes that the other VREfm types.This can be done using the updated E. faecium/E.lactis Virulence-Finder [38].An Australian VRE study found that major strains of E. faecium isolated over 15 years showed unique virulome and resistome profiles with no indication of increasing virulence or antimicrobial resistance determinants.The strains were distantly related and the acquisition of different genes encoding similar antimicrobial resistances suggested the independent evolution of each strain [39].
Our Linezolid is indicated for Gram-positive infections and approved for the treatment of bacterial pneumonia, skin and skin structure infections, and VRE infections, including infections complicated by bacteraemia [41].In Denmark, the consumption of linezolid has increased from 0.48 defined daily doses (DDD) per 100 bed-days in 2015 to 0.65 DDD per 100 bed-days in 2022 [14].Using the LRE-Finder, we detected 45 LVREfm isolates among the 4,052 VREfm and VVEfm isolates; this prevalence was low (1.1%).A low prevalence of LVRE is also seen in Germany [42].Development of linezolid resistance can develop under treatment with linezolid.
We did not have data on linezolid consumption for the individual patients in our study.We investigated the first VRE isolate per 12 months, so it seems likely that the patients were treated with linezolid afterwards.
A recent Danish study identified six patients who initially carried a vancomycin-resistant, linezolid-sensitive Enterococcus, but from whom LVRE closely related to their initial isolate were recovered after linezolid treatment [43].This indicates that the LVRE developed during linezolid treatment.Even though LVRE are rare, they are a concern since only daptomycin and tigecycline can be used for treatment of LVRE infections [44].

Conclusion
The

Funding statement
Part of this work was supported by the Danish Ministry of Health.

Use of artificial intelligence tools
None used.

Figure 1
Figure 1The five healthcare regions and the 10 Departments of Clinical Microbiology and the National Reference Laboratory for Antimicrobial Resistance, Statens Serum Institut, Denmark, 2022
Enterococcus faecium and Enterococcus faecalis van genes per year and isolates, Denmark,2015-2022 (n = 4,862) 2,504 vanA E. faecium, 1,485 vanB E. faecium, 62 vanA/ vanB E. faecium, one vanD E. faecium, 15 vanA E. faecalis and 23 vanB E. faecalis from clinical samples were submitted to SSI (Figure2).We identified additional VRE and VVE isolates in MiBa which had not been submitted to SSI: 59 E. faecalis and 713 E. faecium.These isolates were not genotyped.They are named E. faecium MiBa and E. faecalis MiBa in Figure2.Adding the MiBa isolates to those from the NRL/SSI, a total of 520 VRE and VVE isolates were detected in 2015.This increased to 827 VRE and VVE isolates in 2022 (Figure2).Until 2020, vanA E. faecium were most prevalent.Since 2021, vanB E. faecium has dominated (Figure2).The graph contains isolates submitted to the National Reference Laboratory for Antimicrobial Resistance, Statens Serum Institut (n = 4,090) and unsubmitted isolates from the The Danish Microbiology DataBase (n = 772).The single vanD E. faecium was detected in 2019.Figure 3 shows the number of clinical VRE and VVE for the five Danish regions per year from 2015 to 2022, assigned by testing DCM.The numbers are a combination of data from the NRL/SSI and MiBa.The Capital Region had the highest number of VRE and VVE.From 2019 to 2022, the number of VRE and VVE in the Capital Region decreased.In the same period, VRE increased in the Zealand Region, Central Denmark Region and North Denmark Region.In Supplementary FigureS1, we additionally provide the incidence of VRE and VVE reported as VRE and VVE per 1,000 bed-days.When looking at VRE and VVE in relation to hospital activity during 2022, the Zealand Region had the highest incidence of VRE and VVE per 1,000 bed-days.
Monthly distribution of the seven most prevalent vancomycin-resistant Enterococcus faecium clusters clusters, by region, Denmark, 2015-2022 (n = 4,862) [14]in the country, both in number of bed-days and number of admissions[14].When looking at VRE and VVE in relation to hospital activity in 2022, the Zealand Region had the highest incidence of VRE and VVE per 1,000 bed-days, which was higher than the VRE and VVE incidence in the Capital Region.
[33,34]vanA-vanB E. faecium was first detected in Central Denmark Region in October 2016.This cluster was the only one that did not spread to all Danish regions.The origin of this cluster is unknown and VRE that contain both the vanA and the vanB gene are unusual.Moreover, ST17 vanA/vanB E. faecium isolates have been reported in Vietnam and ST117 vanA/vanB E. faecium isolates in Greece[33,34].

Table
Genetic characterisation of linezolid-and vancomycin-resistant Enterococcus faecium isolates, Denmark 2015-2022 (n =35) [40]y included only clinical VRE and VVE.Whether this distribution is also representative of colonisation with enterococci is unknown.A small study from DCM Hvidovre, Denmark examined whether a patient with a positive VRE clinical sample had the same VREfm in a preceding screening sample (within 60 days).The VREfm pairs (screening isolate and invasive isolate) were sequenced with WGS.Of 19 VREfm pairs, cgMLST types matched in 13, and one additional pair had matching vanA plasmids.Infection was thus preceded by colonisation with the same isolate in 13 of 19 patients[40].
present study highlights the emergence and spread of seven VREfm and VVEfm clusters in Denmark.The spread of the VVE cluster, ST1421-CT1134 vanA E. faecium, in Denmark is of high concern, especially since VVE diagnostics are challenging.Therefore, this cluster is likely to be underdetected, which facilitates further spread.In 2015, the Danish national VRE programme produced WGS data on clinical VRE and VVE isolates.It is costly but allows for further characterisation of isolates for surveillance and outbreak detection and research without additional laboratory work.Currently, cgMLST is being used to follow the spread of VREfm and VVEfm both within the individual hospitals and between hospitals even outside the regions and too other countries.In addition, cgMLST is used for comparison for VRE isolates imported from abroad.We detected only a small proportion of LVRE (1.1%) our VREfm and VVEfm isolates.Nevertheless, this is of concern since the treatment options for LVRE are extremely limited.To reduce further development of LVRE, consumption of linezolid should be kept to a minimum.This study was carried out as part of a task imposed on Statens Serum Institut according to the national legislation as specified in the Danish Health Care Act (Sundhedsloven § 222).The need for ethics approval and informed consent is therefore unnecessary according to national legislation, cf.implementing decree 2020-09-01, number 1338, about scientific regulatory procedure of health science research projects and health data scientific research projects.The manuscript only contains aggregated results and no personal data.The article is therefore not covered by the European General Data Protection Regulation.